The glycolipids such as sulfoquinovosyl diacylglycerol (SQDG), isolated from wild-type cyanobacterium Synecho-cystis sp. PCC 6803, and monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG), ob-tained from whole wheat flour, have been investigated by using fast atom bombardment tandem mass spectrometry (FAB-MS/MS). Collision-induced dissociation (CID) of sodium-adduct molecular ions [M-H+2Na]+ from SQDG and [M+Na]+ from MGDG and DGDG generates ions which are characteristic of the structure of the polar head group as well as the fatty acid composition. In particular, the charge-remote fragmentation along the fatty acid chains observed in the high-mass range was useful to locate the double-bond positions. The major component ion ([M-H+2Na]+ at m/z 865) of SQDG contains oleic and palmitic acids at the sn-1 and sn-2 positions, respectively. The predominant molecular ion ([M+Na]+ at m/z 801) of MGDG contains two linoleic acids. One of the two major species of DGDG contains palmitic and linoleic acids ([M+Na]+ at m/z 939) at the sn-1 and sn-2 positions, while the other contains two linoleic acids ([M+Na]+ at m/z 963). The utility of FAB-MS/MS in the structural determination of glycolipids in mixtures of biological origin has been demonstrated. © 1997 John Wiley & Sons, Ltd.